Light sheet fluorescence microscopy or selective plane illumination microscopy uses a thin sheet of light to illuminate a sample, whilst fluorescent images are taken perpendicular to the illuminated plane. This geometry gives light sheet fluorescence microscopy multiple advantages over other types of microscopy: Firstly, the unilluminated part of the sample remains unexposed to light and cannot be detected. This enhances the axial resolution and image contrast, and also reduces photo-bleaching and phototoxicity to which the sample is exposed. Secondly, the axial resolution of light sheet fluorescence microscopy is mainly determined by the thickness of the light sheet, which is independent of the detection optics. Hence, low magnification objectives can be used for a large field of view, while still achieving good axial resolution. Thirdly, as the whole plane is simultaneously illuminated and imaged, the imaging speed is enhanced compared to scanning confocal microscopy. These advantages make light sheet fluorescence microscopy suitable for constructing 3D images of large samples and even long term monitoring of a living sample. This modality can be extended by utilizing more advanced beam shapes, such as the Bessel beam or the Airy beam.
Present methods for recording 3D stacks of samples in light sheet fluorescence microscopy include either mechanically moving the sample along the detection axis or moving the light sheet and the detection objective along the detection axis at a fixed distance from each other. In both methods, gel is usually used to hold the specimen whilst it is mechanically scanned along the detection axis. The resultant interface between the gel and water introduces optical aberration due to the refractive index mismatch between the two media, thus reducing image quality. In addition, long term monitoring using this mode of confinement can restrict the development of the biological sample. Also, for mobile specimens, such as ocean swimming micro-organisms, the specimen has to be anaesthetized or physically constrained with sufficient force to overcome beating cilia to stop the specimen's movement. The use of anaesthetics and/or physical force may compromise the development and normal functioning of the organism, particularly if required for prolonged periods of time.